Study of the role of Nucleoside Diphosphate (NDP) kinase in the phosphorylation of nucleoside analogs used in anti-viral therapies.

Characterization of the interaction beween human NDP kinase type B and single strand DNA; Role in the regulation of the transcription of the c-myc oncogene.

Biochemical characterization of the protein NEMO, an essential element of the NF-kB signal transduction pathway involved in several human genetical deseases.

Study of the cAMP dependent protein kinase from Dictyosteliumdiscoideum and search for specific inhibitors.

Mechanistic studies on recombination in retrovirus

Mechanistic studies on recombination in retrovirus.

Catalytic mechanism of NDP kinase

Benoit schneider, Sarah gallois-montbrun and Dominique deville-bonne

Nucleoside Diphosphate Kinase (NDP Kinase) catalyzes the transfer of phosphate from a nucleoside triphosphate to a nucleoside diphosphate by a ping-pong mechanism involving the transient phosphorylation of a histidine residue. The 3'-OH group of the ribose of the nucleotide substrate plays a crucial role in the " sustrate assisted " reaction mechanism (Fig. 1).

We study the reaction mechanism by using point mutants of active site residues. Mutants H122G and H122A of Dictyostelium NDP kinase are inactive but they still catalyze the synthesis of imidazole-phosphate from ATP and a exogenous nucleophile as imidazole (collaboration D. Herschlag, Standford, USA). We have studied the ionisation state of Lys 16 and Tyr 56 participating in the phosphotransfer. While both residues interact in the active form of the wild type enzyme, Tyr 56 has an unusual pK in the K16A mutant. However, the substitution of these " catalytic " residues has less effect on the activity than the absence of the 3'-OH group on the sugar moiety of the substrate. We have synthesized a stable analog of the phosphorylated intermediate by chemically modifying the cysteine in the active site of the H122C mutant. This phosphorylated enzyme has an increased affinity for NDPs as compared to NTPs. Dictyostelium NDP kinase has a higher activity than the major human isoforms, NDPK-A and NDPK-B. Comparison of the active sites shows a single difference with a His interacting with the alpha-phosphate in the Dictyostelium protein (His59 in Fig. 1) is substituted by a Leu in the human enzymes. Thus, the mutant L55H of human NDPK-A was purified as a recombinant protein and shown to carry an significantly increased catalytic activity.

In vitro study of the phoshorylation of antiviral nucleotide analogs by NDP kinase

Nucleotide analogs like AZT and d4T are used in all current therapies against AIDS in association with other drugs in multi-therapy protocoles. However they are delivered to the patients as uncharged nucleosides in order to allow cell penetration. Thus they need to be phosphorylated into triphospho-derivatives by cellular kinases before they can act as chain terminator blocking the viral reverse transcriptase. The last step in this activation pathway is thought to be catalyzed by NDP kinase.

We have studied the reactivity of recombinant human NDP kinase with several analogs currently used in clinics (ddI, ddC, AZT, d4T), both at the biochemical and at the structural level (collaboration J. Janin, LEBS, Gif-sur-Yvette, France). We showed that phosphorylation of the analogs is much slower than for their natural counterpart, indicating that NDP kinase may be a limiting step in the activation process. Both kinetic and binding constants were determined. Combining biochemichal and structural data, we have proposed a precise mechanistic model for the phosphorylation of anti-AIDS nucleotide analogs by NDP kinase.

3TC is a L-derivative of thiacytidine recently introduced in therapeutic protocols for AIDS and hepatitis B and beta-L-desoxynucleosides (beta-L-thymidine and beta-L-2'-desoxycytidine) have recently been recognized as putative anti-hepatitis B agents. We have shown that both nucleotides and desoxynucleotides in -L configuration and in particular 3TC are very poor substrates for NDP kinase, (collaboration A. Faraj and J.P. Sommadossi, Novirio-CNRS and Université Montpellier II). This suggests that new yet unidentified kinases may be responsible for the cellular synthesis of the L-XTP and L-dXTP.

Ribavirine, a guanosine analog which is the only nucleoside analog used in clinics against RNA virus, is a good drug candidate against dengue, a viral disease responsible for hemorragic fevers. Ribavirine is active in the cell as a triphospho-derivative. We have screened new derivatives of ribavirin modified on the nucleobase for increased drug efficiency that would allow the use of lower doses and decreased toxicity. However, none of the derivatives showed an increased phosphorylation rate as compared to ribavirine, probably due to the fact that they all lack a free 3'-OH group on the sugar moiety (collaboration L. Mulard, Unité de Chimie Organique, Institut Pasteur et B. Canard, ESIL,CNRS, Marseille).

We have designed new reverse transcriptase inhibitors by modification of currently used nucleoside inhibitors with a borano (BH3) group on the alpha-phosphate (collaboration L. Mulard, Unité de Chimie Organique, Institut Pasteur). The biochemical properties of the borano-derivatives of AZT and d4T were characterized in vitro on NDP kinase and on reverse transcriptase. The X-ray structure of the analog/NDP kinase complex explains the increased reactivity of the borano-derivatives. However, the efficiency of these new compounds at the cellular level is limited by their poor penetration into the cell, due to the electric charge carried by the monophosphate (collaboration with P. Clayette, CEA and C. Périgaud, CNRS-Montpellier). We are currently investigating vectorisation procedures that would allow the prodrug to enter the infected cells.

We have started to modify the active site of human NDP kinase by protein engineering in order to increase its activity for nucleoside analogs. Several mutant proteins were obtained with an increase activity to phosphorylate d4T and AZT as compared to the wild-type human enzymes. The best candidate, combining two mutations in the active site, shows a change in the "specificity factor" of more than 300 as determined on the purified recombinant protein. Current studies are aimed at determining whether the expression of this mutant protein in cultured cells could change their capacity to phosphorylate nucleoside analogs and correspondingly increase their sensitivity to these drugs, in the perspective of cell therapy protocols.

NDPK-B is an isoform of human NDP kinase which is involved in the regulation of the expression of the c-myc oncogene. We showed that NDPK-B binds specifically to single stranded DNA using the purified recombinant NDPK-B and synthetic oligonucleotides which mimic the positive regulation element of the c-myc gene. In order to explore peptidic determinants making specific contacts with the single strand DNA, we developed a new strategy based on the combination of laser-UV irradiation and matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF). The identification of peptides covalently linked after laser-UV irradiation allowed to propose a structural model of the NDPK-B bound to the single stranded DNA (collaboration J. Rossier, Ecole Supérieure de Physique et Chimie Industrielles de la Ville de Paris). This model which shows that the active site acts as a binding template to anchor the oligonucleotide (see Fig. 2), was recently strengthened by mutagenesis as well as by kinetic experiments at the steady state indicating that the C-terminal residue E152 within the active site of NDPK-B is a common determinant for the binding of the substrate and for the interaction with the oligonucleotide.

Using protein permeant cross-linkers which react specifically with cysteine residues, we showed that NDPK-B binding occurs in the nucleus as a hexamer in several cell types. Using a sensitive assay based on fluorescence energy transfer (FRET), we showed that NDPK-B is endowed with unwinding activity since double-strand DNA or stem-loop structures are denatured in the presence of the recombinant protein. Taken together these studies suggest that NDPK-B is an architectural single strand transcription factor which binds to a single strand region contained in triplex or tetraplex DNA structures. This property is not carried by the second isoform of human NDP kinase (NDPK-A) although it shares 88% sequence identity with NDPK-B. The overexpression of NDPK-A in mouse embryonic fibroblasts has a negative dominant effect on the transcription of the c-myc gene. Using permeant cross-linkers, we showed that this effect was due to the in vivo association of NDPK-A subunits with NDPK-B subunits leading to an increase of A/B heterohexamers.

Biochemical studies of the NEMO protein, an essential component of the NF-kB signalling pathway

Fabrice Agou and Stephane Goffinont

NF-kB is an important signal transduction pathway involved in the cell responses to a variety of stimuli including inflammatory processes, stress etc . The cascade controls a series of genes that are transcribed through the activation of an NF-kB transcription factor. NEMO (NF-kB Essential MOdulator) recently discovered in the Unité Biologie Moléculaire de l'Expression Génique (Institut Pasteur), is a protein required for the activation of specific kinases which activate the NF-kB transcription factor. Recently, mutations in NEMO were associated with two human diseases Incontinentia pigmenti and ectodermal dysplasia.

The study of NEMO is performed in close collaboration with G. Courtois and A. Israël (Unité de Biologie Moléculaire de l'Expression Génique, Institut Pasteur). In order to examine biochemical properties, recombinant NEMO was produced in E. coli and purified in the presence of non-ionic detergents to avoid the formation of protein aggregates. The recombinant protein copurifies with the E. coli DnaK (Hsp 70) suggesting a role for chaperon proteins in the formation of oligomeric complexes of NEMO in vivo. The quaternary structure of NEMO was studied by sedimentation velocity and by equilibrium sedimentation. Cloning an purification of a C-terminal fragment allowed to show that the association of the native protein in dimers and trimers is due to a specific sequence forming coiled coils structures. The presence of oligomeric forms of NEMO has also been investigated in vivo on cultured cells. Using a cell permeable bifunctional crosslinking reagent, the presence of dimeric and trimeric forms was demonstrated. Current experiments are aimed at determining which functional properties of NEMO are altered by the mutations found in human patients.

The Dictyostelium cAMP dependent proteine kinase: search for specific inhibitorsFrançois TraincarDDictyosteliumdiscoideum amoebaeenter a development cycle in response to food starvation. After a few hours, the cells form streams and migrate in response to cAMP signals to form a multicellular aggregate. In a second phase, the latter develops into a fruting body formed of a stalk supporting a mass of spores.

We have previously investigated the role of cAMP dependent protein kinase (PKA) in Dictyostelium development and we have characterized the biochemical properties of the R (regulatory) and C (catalytic) subunits. We have shown that the substitution of the last amino-acids of the C-terminal extension of the catalytic subunit results in a severe loss of catalytic activity.We have discovered a new inhibitory site of AGC (PKA, PKG, PKC) protein kinases. This site could also be present on members of other protein kinase families. PKA could be a therapeutic target against pathogens such as Plasmodium falciparum. Indeed, we have demonstrated the presence of a PKA activity which is necessary for parasite maturation in infected erythrocytes (collaboration G. Langsley, Unité de Signalisation Immunoparasitaire, Institut Pasteur).

We have started to use Dictyostelium as a model system for practicals in primary school and highschool classes. General concepts like cellular communication, differenciation and development can be appropriately illustrated using Dictyostelium in biology teaching.Mechanisms of retroviral recombinationAbdeladim Moumen, Lucette POLOMACK and Matteo NegronI

Homologous recombination is a major source of genetic variability in retroviruses. During their extracellular life retroviruses store genetic information as a single RNA molecule, which is present in two copies within each viral particle. Genetic recombination occurs in retroviruses mostly through template switching during reverse transcription between these two copies of genomic RNA, a process known as "copy choice" or strand transfer. The impact of recombination on the dynamics of retroviral infections has been dramatically illustrated in the case of the spreading of the AIDS pandemic. In this case at least 10% of the infectious strains of HIV originate through recombination among different viral subtypes.

We study the mechanism of retroviral recombination in a reconstituted system, using purified nucleic acids and proteins. We have focused our attention on the role of the structures of the genomic RNA and of a major co-factor of the reverse transcription process, the nucleocapsid protein (NC), an RNA chaperone known to enhance strand transfer in vitro. Our results indicate that the structure of the genomic RNA is the main determinant in the recombination process, in contrast to the current opinion of a copy choice process governed by pausing of reverse transcription (Fig. 3). In order to dissect the molecular mechanism of recombination, we are currently trying to identify the structural features of the genomic RNAs that characterise a recombination hot spot. In parallel we are developing a system to study retroviral recombination in infected cells in culture (collaboration P. Charneau, Unité de Virologie Moléculaire et Vectorologie, Institut Pasteur). Other projects aimed to evaluate the similarities between the mechanism of recombination in retroviruses and in other viruses are ongoing.

Figure 1 : The active site of NDP kinase

The Figure shows a dTDP molecule bound in the active site of Dictyostelium NDP kinase.The catalytic histidine (H122) is shown, as well as several residues important for binding of the substrate and catalysis. The crucial hydrogen bond between the ribose 3'OH and the O7 of the beta-phosphate is also shown.

Figure 2 : Modèle du complexe NDPK-B/Oligonucleotide

Oligonuclmeotide binding to NDPK-B

Figure 3 : A model for recombinaison by HIV reverse transcriptase

A possible pre-recombination intermediate. Top view of the catalytic subunit of the reverse transcriptase from HIV-1. Since the enzyme resembles a right hand, its different subdomains have been called, fingers, palm, and thumb. In red is shown the RNase H domain, responsible for the degradation of the RNA template once copied at the polymerase active site. The enzyme is drawn in the process of polymerising on the donor template. The acceptor template is hybridised to the nascent DNA behind the RNase H site. The transient path followed by the acceptor RNA (drawn arbitrarily) is meant to rise above the plane of the drawing. The folding adopted by the acceptor RNA is considered, in our working hypothesis, as critical for displacing the donor template from the polymerase active site. The mechanism by which this displacement occurs is currently under investigation in the laboratory.